Analysis of metabolic activity in cells using extracellular flux rate measurements

Abstract

Disclosed are methods for non-destructively measuring in vitro the effect on cellular metabolism of the addition to animal cells in culture of a soluble molecule potentially capable of perturbing the biological state of the cells, such as a drug or drug candidate, a toxin, a ligand known or suspected to bind to a cell surface receptor, a nutrient, a cytokine, a growth factor, a chemokine, a metabolism inhibitor or stimulator. Also disclosed are methods for measuring cell viability, vitality, or quality, e.g., in anticipation of the execution of an experiment on the cells. The measurements are done by observing alteration in the rates of consumption or production of extracellular solutes related to aerobic and anaerobic cellular metabolism, such as oxygen, protons, nutrients, carbon dioxide, lactate, or lactic acid. The methods are particularly useful in drug discovery efforts, such as cancer drug discovery and searches for modulators of cellular metabolism.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of and priority to U.S. Provisional Patent Application Ser. No. 60 / 724,669, filed Oct. 7, 2005, and is a continuation-in-part application of copending U.S. patent application Ser. No. 10 / 688,791, filed Oct. 17, 2003, entitled “Method and device for measuring multiple physiological properties of cells,” and published as US / 2005 / 0054028, on Mar. 10, 2005, and copending U.S. patent application Ser. No. 11 / 486,440, filed Jul. 13, 2006, and entitled “Cell analysis apparatus and method.” The entire disclosures of each of these applications are incorporated by reference herein for all purposes.FIELD OF THE INVENTION [0002] This invention relates to cell analysis methods, and more particularly to methods for probing animal cells, tissues and cellular organelles, such as mammalian cells in culture by measuring alterations in their metabolism, e.g., upon exposure to an environmental stress or a chemical such as...

AI Technical Summary

Benefits of technology

[0019] In yet another embodiment, the invention provides a method for analysis of cell culture quality comprising the steps of measuring in a cell medium the rate of change in concentration of both an extracellular solute which is a component of cellular aerobic metabolism and an extracellular solute which is a component of cellular anaerobic metabolism, and comparing the measured rates of change to a standard informative of known cell culture respiration rates, thereby to assess the respiratory capacity of the culture as a measure of cell vitality and cell quality. This cell quality measurement method may comprise comparing the measured rates of change to rates measured in a culture comprising a known number of healthy cells of the same cell type or of a cell type having comparable metabolic characteristics to the cells under quality assessment. Preferably, this method comprises seeding cells at a predetermined density in a test well prior to the measuring step thereby to enable direct comparison of the measured rates of change to a standard. This method does much more than take a measurement indicative of whether the cells in a culture are alive, as it can measure metabolic rate; measure relative contribution of aerobic (oxidative phosphorylation) versus anaerobic (glycolysis) processes for generation of ATP; measure adherent cells in a microplate; or measure suspended cells in a microplate. Furthermore, the quality assessment is non destructive, and therefore the planned experiment on the cells can be conducted after assessing cell vitality and quality.

[0020] In a related aspect, the invention permits the scientist to obtain data indicative of respiratory (or metabolic) capacity of a cell culture without cell counting. This is done by measuring a basal metabolic rate or rates (i.e., rates of change of OCR, ECAR etc.), before the addition of any metabolism altering substance, followed by adding to the culture a drug that increases metabolism, and then repeating the measurement. A class of substances suitable for this purpose are drugs known to uncouple the TCA cycle within a cell, thereby producing waste heat in lieu of providing energy via ADP to ATP conversion. The increased respiration rate is indicative directly of metabolic capacity, and the ratio can be used as such a measure independent of the actual amounts of cells in the test well in which the measurements were made. This eliminates the need for cell count to normalize data, and can be particularly valuable when cell number is different in various wells or when cells proliferate during the experiment (particularly cancer cells).

Problems solved by technology

Indeed, in a living cell there are thousands of chemical reactions, each coupled and progressing via a complicated network of inter and extra cellular processes.

In fact, the complicated aerobic process for metabolism of nutrients, involving multiple steps of substrate conversion, the TCA cycle, and electron transport chain, is probably one of the most fragile functions performed by a cell.

Unfortunately, few methods exist to measure the metabolic properties of mammalian cells.

Neither system was able to produce data of sufficient quality to analyze drug-induced metabolic behavior changes within a typical effective dose range.

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